Embryonic/Postnatal Mouse Neuron Culture Protocol

Drop 12 mm coverslips into a 200 ml beaker containing 95% Ethanol (or methanol). (Leave at least 0h 1m 0s).

Plate coverslips on a 24-well plate and allow to completely dry (>0h 30m 0s).

Dilute PDL to 100 with borate buffer (2.5mL PDL solution + 47.5mL borate buffer) and coat coverslips with 0.5mL PDL for 2h 0m 0s in the incubator or 2h 0m 0s in the TC hood. Plastic plates can be coated similarly (100 µL for 96-well).

Wash coverslips or plates 3-5x in tissue culture water. Remove all culture water from coverslips or plates, then allow to dry for 1h 0m 0s + in TC hood.

Add neuron medium to plates and incubate at 37°C in incubator > 1h 0m 0s to equilibrate (50 µL for 96-well, 0.5 mL 24-well, 1 mL 12-well, 2 mL 6-well). If using a 96-well plate, add 200µL of PBS in periphery wells and 150µL of PBS between wells.

Equilibrate a T25 or T75 flask of neuronal medium in the incubator. Can also warm Neurobasal to Room temperature for wash steps.

Make the papain dissociation solution (see Media section). Each T25 flask can hold tissue from 1 to maximum 20 mouse preps. Sterile filter (syringe filter or Steriflip, depending on volume) and keep in T25 flask in the incubator.

Apply CO2 to dam as indicated in IACUC protocol.

Dislocate spine.

Pin dam, abdomen up, to Styrofoam covered with lab mat.

Spray dam with 70% ethanol thoroughly.

With dissection scissors, open the abdominal cavity, avoiding internal organs.

Grasp the uterus with forceps. Lift and cut the uterus out. Transfer to a 10 cm culture dish.

Cut the uterus and remove the embryonic sacs to release embryos into a fresh dish On ice.

Decapitate pup, allowing head to fall into a 10 cm dish filled with HBSS On ice.

Using forceps, hold head by the eyes. Using the other hand and a curved or angled forceps, pinch the scalp just behind the eyes and pull back, tearing it off.

Next, use a micro-scissor to cut down the mid-sagittal skull, and gently peel the skull off with forceps, being careful in case any tissue still connects it to the rest of the skull. Gently remove brain (using a spatula or curved forceps) and place into Petri dish filled with ice cold sterile Hibernate Medium.

Gently separate hemispheres, removing the thalamus, striatum, and brainstem.

Grasp the olfactory bulb and pull caudal, slowly removing the meninges from the cortex. While still grasping the meninges, flip the hemisphere and remove meninges from the hippocampus.

Using micro-scissors, cut the hippocampus away from the cortex.

Hippocampi can be left whole. If cortical tissue is to be used, it should be cut into 1 mm³ segments.

Place all segments into a conical with Hibernate medium.

Bring to biosafety cabinet. Everything from here on should be done in a sterile biosafety cabinet. Pipette out Hibernate medium and rinse twice with fresh, Room temperature HBSS.

Use serological pipet to transfer chunks to papain and move to 37°C incubator, laid flat.

Digest for approximately 0h 5m 0s-0h 20m 0s or until chunks have fallen apart and look something like cotton candy.

Add 50µL [170 U] DNase/10mL papain solution and gently rotate until pieces of tissue are separated.

Use 10 mL serological pipet to move tissue from T25 flask into 15 mL conical.

Rinse chunks 2-3 times using 5 ml Neurobasal (warmed to 37°C).

Remove as much media as possible and resuspend cells in 2mL of neuron medium.

Mechanically dissociate cells (do not over triturate!). First, pipet up and down with 1000 µL tips ~10-15 times (when done properly the medium becomes slightly opalescent with dissociated cells). Next, use a 200 µL tip ~20 times. The chunks should now be completely dissociated into individual cells.

Bring total volume to 6 mL Neurobasal and strain through a 40 μm cell strainer into a 50 mL conical, rinsing strainer with 2 mL Neurobasal before and after. Transfer cells to 15 mL conical.

Centrifuge cells at 1000x g. Resuspend the pellet in 2 mL of neuron media, mix and count.

Dilute cell suspension to 1,000,000 cells/mL. For 96-well, cells should be diluted to 170,000 cell/mL.

Add the appropriate volume to the well of the neuron media-containing dishes so that each well contains:

• 17,000 cells (96-well)

  Note

  For 96-well plates cells should be gently agitated in a reservoir before being added directly to the middle of the well.

• 100,000 cells (24-well)

• 250,000 cells (12-well)

• 1,000,000 cells (6-well)

Gently agitate plates back and forth in each direction to spread cells. Place in incubator.

Cells can be checked the next day for adherence and even distribution. Neurons will start to sprout neurites within the first few days.

Add additional media to each well once a week:

• 20 μL (96-well)
• 115 μL (24-well)
• 330 μL(12-well)
• 1 mL (6-well)